Glia maturation factor (hereinafter GMF) is a protein, found in the brain of many animals, including man, which is capable of regulating the growth, development and differentiation of cells of neural origin (neurons and glia) and possibly other cell types. The term "glia maturation factor" (GMF), as used herein, includes both the crude preparations of GMF and pure GMF, whereas the term "glia maturation factor" (hereinafter GMF), as used herein, specifically denotes pure homogeneous GMF protein identifiable by a defined amino acid sequence.
GMF was discovered by Ramon Lim in 1972. Over the years, several biological functions have been identified for GMF, suggesting GMF as a potential therapeutic agent for a number of diseases or pathologic conditions in humans and animals. These functions are summarized as follows:
(I) Evidence that GMF can enhance nervous system regeneration. Lim & Miller (Experientia, 41: 412-415 (1985)) have shown that in newborn rats sustaining brain injury, GMF treatment prevents atrophy of the brain. In another instances, Lim, Miller & Toffano (Trans. Am. Soc. Neurochem., 16: 307 (1985)) reported that when rat brains are transacted in the nigro-striatal region, injection of GMF into the brain helps the recovery as monitored by the neuron-specific enzyme tyrosine hydroxylase. Palatucci et al. (Soc. Neurosci. Abstr., 14: 584 (1988)) demonstrated that in rats sustaining caudate lesion in the brain, treatment with GMF enhances recovery from behavioural deficit. Nieto-Sampedro et al. (Neurosci. Letters, 86: 361-365 (1988)) demonstrated that there is an enhanced release of GMF from the injury site in the brain. Lim et al. (Trans. Am. Soc. Neurochem., 19: 83 (1988)) demonstrated that there is increased production of GMF in the satellite cells surrounding the sciatic nerve after the nerve is cut. The above suggests that GMF is involved in the regeneration of the nervous system. Thus, GMF is a potentially useful therapeutic agent for injuries to the brain, spinal cord and the nerves.
(2) Evidence that GMF can enhance nervous system development. The fact that GMF promotes regeneration of the nervous system suggests the possibility that it can promote the development of the nervous system, since development and regeneration involve similar mechanisms. Other evidence supports a role for GMF in development. For example, using a monoclonal antibody designated G2-09 specifically directed toward GMF, Lim et al. (Dev. Brain Res., 33: 93-100 (1987)) found that the level of GMF is highest in embryonic brain. In tissue culture, GMF can stimulate the differentiation of astrocytes (Lim et al., Science, 195: 195-196 (1977)) and Schwann cells (Bosch et al., Brain Res., 304: 311-319 (1984)). Lim et al. (Trans. Am. Soc. Neurochem., 16: 307 (1985)) observed that when neurons are isolated from the mesencephalon region of the brain and grown in culture, GMF can stimulate them to take up neurotransmitters, a function characteristic of mature neurons.
GMF helps in the survival of neurons isolated from the cerebellum region of the brain (Guo & Lim, unpublished data). Lim, Miller & Zaheer (Proc. Nat'l. Acad. Sci. USA 86:3901-05 (1989)) found that GMF promotes the differentiation of neuronal tumors by causing them to grow out cell processes (neurites) while at the same time suppressing their proliferation (FIG. 1). Currently, there is not satisfactory treatment for children with abnormal development of the nervous system. Such pathologic conditions often lead to impairment of mental, behavioral or motor activities. GMF can potentially correct such developmental problems.
(3) Evidence that GMF can arrest or reverse the progress of nervous system degeneration. It is known that many neurological diseases are due to premature or abnormal degeneration of certain areas of the nervous system. Such pathologic conditions include Parkinson's disease and Alzheimer's disease and are difficult to treat. Although GMF has not been tested for these neurologic problems, it is conceivable that GMF may help in arresting or reversing the progression of such degenerative processes, given its regulatory role in brain cell development.
(4) Evidence that GMF can arrest or reverse the progress of tumors. It has been documented that GMF can suppress the growth of tumor cells derived from Schwann cells (Lim et al., Proc. Natl. Acad. Sci., 78: 4373-4377 (1981)) and from astrocytes (Lim et al., Cancer Res., 46: 5241.varies.5247 (1986)). Lim, Miller and Zaheer (Proc. Nat'l. Acad. Sci. USA 86:3901-05 (1989)) found that GMF also causes growth arrest in tumor cells of neuronal origin (FIG. 1). While it is clear that GMF exhibits an anticancer effect on brain tumors, it remains possible that the effect may extend to other types of tumors found elsewhere in the body, provided that the tumors develop receptors for GMF and are thus responsive to it. GMF could be an excellent therapeutic agent for all these tumors, not only as a direct growth suppressive agent, but also as a carrier for tumor destructive agents to reach the cancer site.
In order for GMF to be therapeutically useful, it must be purified to homogeneity, free of other proteins or compounds that may interfere with the action of the agent or cause untoward reactions on the recipient person or animal. GMF obtained by former purification procedure devised by Lim et al. (Biochemistry, 24: 8070-8074 (1985)), although appearing as one band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), did not turn out to be homogeneous and no amino acid sequence was able to be obtained. In the current invention, important changes were made on the procedure. Such changes led to the production of a pure homogeneous GMF protein, called GMF, which exhibits a definitive amino acid sequence. The sequence enabled us to clone the cDNA from a human source. From the cDNA we are able to produce recombinant GMF free of other proteins of human origin in large quantities using genetic engineering technology. For human application, GMF of the human type is preferred to that of other species because of possible immune reaction due to species difference.
Alternative ways to purify GMF may be possible, such as the use of affinity chromatography utilizing antibodies against GMF, or using specific lectins if GMF binds to such substances. However, none of these will be comparable in efficiency to the recombinant DNA method. Nevertheless, it is conceivable that GMF produced by the recombinant method may need to incorporate one or several of these other methods in its final purification.